Klinisk institutt 2

Midtveisevaluering - Jan Inge Bjune



Genomic and epigenomic regulation of adipocyte browning by Irx3 and Irx5

Our group has previously identified Irx3 and Irx5 to be differentially expressed after bariatric surgery in extremely obese patients. Furthermore, these genes were found to be regulated by an enhancer in intron 1 of the FTO gene that contains the strongest known association with obesity in GWAS studies. We identified the causal SNP and found the risk allele to increase the expression levels of IRX3 and IRX5, which in turn lead to repression of mitochondrial thermogenesis and increase in lipid storage.

We therefore made Irx5 knock-out mice and challenged them with a high-fat diet. Irx5 -/- mice exhibited extensive fat loss and resistance to diet-induced obesity. Microarray analysis of the white adipose tissue from the mice was thus performed and top-scoring genes were verifying by RT-qPCR. In addition, we have found similar genes to be differentially expressed in white adipose tissue from obese and lean patients. To distinguish effects directly attributed to Irx5 ablation from secondary effects resulting from fat loss, we have performed siRNA knock-down of Irx5 through differentiation of the white adipocyte cell line iWAT2 followed by RNA-sequencing. To address the question of whether Irx5 directly regulates the affected genes, we have performed transactivation essays and we are currently doing gel shift (EMSA) experiments. We have identified Amyloid beta A4 precursor protein (App) as a possible mediator of the anti-obesogenic effect of Irx5 ablation

In parallel, we have performed Yeast-2-Hybrid analysis of the IRX5 protein and identified several putative interaction partners. We have verified the interaction between IRX5 and one of the candidates, GBAS in vitro. In addition, subcellular fractions isolated from beige adipocytes through different stages of differentiation were analyzed by Western Blot. Furthermore, for a more in vivo like assessment, immunocytochemistry was performed. Although WB experiments suggested co- localization of Irx5 and Gbas in the same compartment, interactions could not be detected in the selected cell line with ICC. Other cell lines will be investigated. A similar approach is planned for other putative interaction partners that are of particular interest due to their role in epigenetic regulation.

The third project focuses on the identifying the direct target genes of Irx5 during differentiation of the ME3 mouse beige preadipocyte cell line. Several differentiation-timelapses have been performed in which cells have been harvested at day 0, 1, 2, 4 and 7 of differentiation. For each timepoint, cells were treated with a β3-adrenergic agonist for 4 hours before harvest. Samples from each timepoint are currently being subjected to ChIP-seq (against Irx5), open chromatin profiling (ATAC-seq) and RNA-seq for a robust identification of direct Irx5 target genes.